JP2017029137A - Polysensing bioelectronic test plate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test plate for analyzing substances and a system related to such test plate.SOLUTION: Provided is a device which includes: a test plate comprising plurality of wells, each of the wells being configured to contain a substance to be analyzed; a plurality of sensors that are arranged to sense characteristics of the substance, to generate sensor signals based on the sensed characteristics over time, and to associate with each of the wells, at least one of the plurality of sensors sensing a characteristic of the substance that is different from a characteristic sensed by another sensor of the plurality of sensors; and a sensor select circuitry that is arranged on a backplane disposed along the test plate, coupled to the sensors, and configured to enable the sensor signals of selected sensors to be accessed at a data output of the backplane.SELECTED DRAWING: None

Description

  The present disclosure relates generally to test plates for analyzing materials, and systems and methods associated with such test plates.

  Many applications in biology, medicine and toxicology involve real-time detection of complex biophysical, biochemical and functional characteristics of cells in a physiologically relevant 3D environment. These characteristics may change in a non-uniform and temporary manner during progression from a normal state to a disease state and / or when exposed to drugs and toxicants, so that It is desirable to monitor (in a single state or in a colony state).

  One embodiment relates to an electronic test plate having a plurality of wells, each well configured to contain a substance to be analyzed. The sensor is configured to detect a characteristic of the substance and create a sensor signal based on the detected characteristic. The sensors are arranged so that multiple sensors are associated with each well. At least one sensor of the plurality of sensors detects a characteristic of the substance that is different from a characteristic detected by another sensor of the plurality of sensors. The sensor selection circuit is connected to the sensor. The sensor selection circuit is arranged on a back surface arranged along the test plate. A sensor selection circuit allows the sensor signal of the selected sensor to access the back data output. According to certain embodiments, the electronic test plate is optically clear or has an optically clear area that can optically interrogate each well.

  According to one embodiment, the electronic test plate comprises a test plate having a plurality of wells, each well configured to contain a substance to be analyzed. The sensor of the electronic test plate is configured to detect a characteristic of the substance and generate a sensor signal based on the detected characteristic. The sensors are arranged so that multiple sensors are associated with each well. At least one sensor of the plurality of sensors is configured to detect a characteristic of the substance that is different from a characteristic detected by another sensor of the plurality of sensors. A sensor selection circuit of an electronic test plate arranged on a back surface extending along the test plate connects to the sensor. The sensor selection circuit allows the sensor signal of the selected sensor to be accessed with the back data output. The lead-out circuit receives and processes the selected sensor signal present in the data output.

  Certain embodiments relate to a method for manufacturing an electronic test plate. The method includes creating a test plate having a plurality of wells, each well configured to contain a substance to be analyzed. An electronic circuit comprising a plurality of sensors and a sensor selection circuit connected to the sensors is manufactured. The plurality of sensors are configured to detect a feature of the substance and create a sensor signal based on the detected feature. The sensor selection circuit allows access to the sensor signal of the selected sensor with the back data output. The sensors are aligned with respect to the well such that multiple sensors are associated with each well. The plurality of sensors associated with the wells are each configured to detect a characteristic of the substance that is different from a characteristic detected by another sensor of the plurality of sensors.

  Certain embodiments include a method comprising detecting a plurality of characteristics of an analyte to be analyzed disposed in a well of a test plate. The plurality of features is detected using a plurality of sensors associated with each well. At least one of the plurality of sensors is configured to detect a characteristic of the material that is different from a characteristic detected by another sensor of the plurality of sensors. The sensor signal is created based on features detected over time. The address line is activated so that the sensor signal of the selected sensor can be accessed by data output.

  The following description refers to the following drawings, in which like reference numerals may be used to identify similar / same elements in multiple drawings. Unless otherwise indicated, the drawings are not necessarily drawn to scale.

FIG. 1A is a top view of an electronic test plate according to an embodiment. FIG. 1B shows a top view of a section of the test plate of FIG. 1A. FIG. 1C shows a cross-sectional view of a test well and a plurality of sensors associated with the test well. FIG. 1D is a simplified perspective view of some layers of the electronic test plate of FIG. 1A. FIG. 2A is a top view of a portion of a back surface in the vicinity of a pixel with four subpixel sensors according to some embodiments. FIG. 2B is a side cross-sectional view of the test well and a portion of the back of the test plate. FIG. 2C is a top view of nine pixels, where one pixel may be associated with one test well that includes an oxygen sensor according to some embodiments. FIG. 2D provides a top view of the oxygen sensor of FIG. 2C. FIG. 2E is a cross-sectional view illustrating a pixel including an oxygen sensor according to an embodiment. FIG. 2F is a top view of a portion of a test plate with the test well of FIG. 2B showing nine pixels according to an embodiment. FIG. 2G shows a top view of the back and test plates having a test well pitch larger than the back pixel pitch. FIG. 2H shows a back surface having a pixel pitch equal to the test well pitch. FIG. 3A is a photograph of a prototype of an electronic test plate fabricated using different well sizes and materials according to various embodiments. FIG. 3B is a photograph of a prototype of an electronic test plate fabricated using different well sizes and materials according to various embodiments. FIG. 3C is a photograph of a prototype of an electronic test plate fabricated using different well sizes and materials according to various embodiments. FIG. 3D is a photograph of a prototype of an electronic test plate fabricated using different well sizes and materials according to various embodiments. FIG. 3E shows an exemplary 3D culture of MDA-MB-231 cancer cells on a glass slide coated with thin gold. FIG. 3F shows an exemplary 3D culture of MDA-MB-231 cancer cells on silicon. FIG. 4A is a schematic diagram of an exemplary TFT sub-pixel sensing circuit and sensor selection circuit that can be implemented to provide sensor signals from impedance sensors, pH sensors, optical sensors, acoustic sensors, according to an embodiment. give. FIG. 4B shows a configuration for providing incident light for optical detection in various embodiments. FIG. 4C illustrates a configuration for providing incident light for optical detection in various embodiments. FIG. 4D illustrates a configuration for providing incident light for optical detection in various embodiments. FIG. 5A is a block diagram of an electronic test plate 500 according to some embodiments. FIG. 5B is a block diagram of an electronic test plate similar in some respects to the electronic test plate of FIG. 5A, including optional features according to certain embodiments. FIG. 6 is a block diagram of a test system that incorporates one or more of the electronic test plates according to embodiments described herein. FIG. 7 is a flow diagram illustrating a method using a bioelectronic test plate and data output process according to an embodiment.

  Embodiments disclosed herein include a multi-sensing electronic test plate comprising a test plate having a plurality of test wells and a plurality of sensors associated with each test well. The plurality of sensors detect characteristics of the substance to be analyzed disposed in the test well, and create a sensor signal based on the detected characteristics. At least one of the plurality of sensors associated with the test well can detect a characteristic of the substance that is different from a characteristic detected by another of the plurality of sensors. In certain embodiments, one or more of the sensors may be configured to detect material features in multiple dimensions (eg, 2D detection or 3D detection). The sensor selection circuit can access the sensor signal of the selected sensor at the data output of the electronic test plate. In some embodiments, the sensor selection circuit may comprise a thin film transistor (TFT) switch disposed on the back surface that is driven by a selection line to access the sensor output.

  In certain implementations, each test well can be connected to an electronic test plate as disclosed herein by other methods such as mass spectroscopy, atomic force microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, and scanning electron microscopy. In addition to the type of analysis, it can be used in combination with various types of optical analysis techniques that interrogate living or fixed cells and tissues (eg, optical microscopy, spectral analysis, fluorescence tagging analysis) Having at least one optically transparent coupling area to allow Optical analysis techniques may include both label-free techniques and label-specific techniques. In certain embodiments, these analytical techniques can be used to assist and / or confirm results achieved using multiple sensors of an electronic test plate. The electronic test plate described in the embodiments herein may be suitable for use with a variety of analytical techniques in addition to multiple sensing by multiple sensors. Additional analytical techniques that may be useful when combined with the disclosed multi-detection electronic test plates are: Veiseh, Mandana et al., “Guided cell patterning on gold-dioxide substratates by surface moleculare 25”, Bioengineering 25, Bioengineering 25 -3324, Veiseh, Mandana et al., “Effect of silicon oxidation on long-term cell selectivity of cell-patterned Au / SiO2 platforms”, J. Am. AM. CHEM. SOC. 128 (2006), 1197-1203, and Veiseh, Mandana et al., “Single-cell-based sensors and synchrotron FTIR spectroscopy: A hybrid system 3-27”.

  The embodiments disclosed herein can also be used to bridge large gaps in the detection of physiologically relevant cell lines for applications in biology, medicine and environmental toxicology. These techniques are for in vivo microenvironment, personalized medicine, and biological and clinical evaluation of heterogeneity in an environment that replicates aspects of specimen screening in the healthcare and toxicology fields. Give a new tool. They are also detected by new multiple biomarkers and temporal / spatial correlations that are not successful by static, label-specific measurements on fixed cells, or by separate devices or at different times 1 The sum of the types of biomarkers will be revealed. Because different biomarkers can be monitored simultaneously and continuously and can be multiplexed with large numbers of parallel cell sensor arrays, orders of much more data can be collected than existing approaches. This increases the likelihood that the machine-learned algorithm will identify low-occurrence non-uniformities, new temporal signals, or phenotypic patterns. Comparison of adjacent wells that differ in only one aspect can provide differential information that allows a high level of common mode noise rejection.

  1A is a top view of an electronic test plate 100 according to one embodiment, FIG. 1B shows a top view of a section of the test plate 100, and FIG. 1C is cut along the AA ′ plane. A cross-sectional view of a test well and a plurality of sensors associated with the test well is shown, and FIG. 1D is a perspective view of a portion of an electronic test plate.

  The electronic test plate 100 has a plurality of test wells 111A-114A defined by well walls 160 extending in the z direction below the upper surface 101 of the test plate 100. The test wells 111A-114A shown in FIG. 1A may be arranged in various patterns and / or sizes (eg, diameter and / or depth) as shown in FIG. 1A. As shown, the test plate 100 has four compartments 111-114, each compartment having test wells 111A-114A, and the diameter of the test wells 111A-114A varies from compartment to compartment. In one particular example, the diameter of the test well 111A in the compartment 111 of the test plate 100 is 5 mm in diameter at the surface 101 of the test plate 100, and the diameter of the test well 112A in the compartment 112 of the test plate 100 is The diameter of the test well 113A in the surface 113 of the test plate 100 in the section 113 of the test plate 100 and in the section 113 of the test plate 100 is 1 mm in diameter in the section 101 of the test plate 100 and the test well 114A in the section 114 of the test plate 100. Is 1 mm in diameter on the surface 101 of the test plate 100. The test wells 114 </ b> A are arranged in a denser pattern (a larger number of test wells per area) than the pattern of the section 113. It will be appreciated that the above example is just one of many configurations of test wells in a test plate. Further, although test plates with different sizes and patterns of test wells (eg, rectangular or triangular) are possible, in many applications, the test plate includes equally spaced test wells and / or Each test well is the same size and has the same diameter.

  A plurality of sensors 120 are associated with each test well 111A-114A. A plurality of sensors 120 associated with the test wells 111A-114A detect a plurality of characteristics of the substance in the test well. Each sensor is configured to detect a feature of a substance in one, two, or three dimensions. For example, one or more of the sensors 120 may be configured to sense a feature of the material across at least a portion of the test well along the x direction, and the one or more sensors may further be along the y direction. And may be configured to detect a feature of the material across at least a portion of the test well, the one or more sensors further detecting a feature of the material across at least a portion of the test well along the z-direction. It may be configured to perform one-dimensional, two-dimensional, or three-dimensional detection over time.

  The sensors 120 associated with each test well 111A-114A may be arranged in a set of different types of sensors, each set of sensors being referred to herein as a “pixel”. Each sensor in a pixel is referred to herein as a “subpixel”. Each sensing sub-pixel of a pixel may be configured to measure a characteristic that is different from a characteristic of the material detected by another sensing sub-pixel of that pixel. The terms “pixel” and “subpixel” are borrowed from a display or imaging device, in which case “pixel” is a detection unit (“subpixel”) that can be repeated in a 2D array. It will be understood to describe a combination of unit cells).

  Sensor pixels and sub-pixels are equally spaced on the back surface, regardless of whether the test plate has large (eg, 5 mm diameter) test wells or small (eg, 1 mm) test wells. You may have the pitch of. More pixels may be associated with each larger test well as compared to the number of pixels associated with the small test well. In some embodiments, the pixel pitch may be, for example, on the order of about 300 μm. Pixel pitches of less than 300 μm are possible, for example, in some implementations a pixel pitch of about 90 μm, or even about 60 μm.

  The achievable number of pixels per test well and / or the number of subpixels per pixel varies depending on the size of the test well and / or the dimensions of the plate. FIG. 1A shows a test plate with a width of 25 mm along the x-axis and a length of 75 mm along the y-axis, but other test plates such as standard culture plates (eg, 127.7 mm × 85.5 mm). Dimensions are also possible. In the first example structure, a 25 mm × 75 mm test plate comprises 15 equally spaced test wells having a circular xy cross section with a diameter of 5 mm (as indicated by section 111 of test plate 100). And 218 pixels may be associated with each test well. In a second example, a 25 mm × 75 mm test plate may have 50 equally spaced circular test wells (as indicated by section 112 of test plate 100), each test well being The diameter may be 3.1 mm and 80 pixels may be associated with each test well. In a third example, a 25 mm x 75 mm test plate may have 288 square test wells (arranged in the pattern indicated by the sections 113 of the test plate 100), each test well being The length along the y-axis is 1 mm, the width along the x-axis is 1 mm, and 10 pixels may be associated with each test well. In a fourth example, a 25 mm × 75 mm test plate may have 592 equally spaced square test wells (as indicated by section 114 of test plate 100), each test well being The length and width may be 1 mm and 10 pixels may be associated with each test well. In the first example, assuming that each pixel contains 4 sensing subpixels per pixel, its back side contains 3,270 pixels, 13,080 sensors, In the second example, the back surface includes 4,000 pixels and 16,000 sensors, and in the third example, the back surface includes 2,880 pixels and 11,520 sensors. In the fourth example, the back surface includes 5,920 pixels and 23,680 sensors. These values are merely examples, and the back surface may contain more or fewer pixels and subpixels. In certain embodiments, the test plate may be configured to provide approximately 40,000 sensors and provide 40,000 unique measurements.

  In some applications, the distance S between wells may be about 5% of the diameter (or length or width) of the test well. For example, in the case of equally spaced square test wells with a width and length of 1 mm, the test wells may be spaced 20% (0.2 mm) of the test well width along the x-axis, y A 20% (0.2 mm) space of the length of the test well may be opened along the axis.

  The electronic test well may be smaller or larger than the 25 mm × 75 mm test plate shown in FIG. According to certain embodiments described herein, an electronic test well includes integrated thin film transistor (TFT) based electronics and active matrix electronics addressing and connection to a large number of sensors at low cost And allows large area glass slides for cell culture well plate arrays. Integrated multiple arrays allow for many unique measurements per slide (eg, 4 measurements per pixel) with few addressing pads and can be easily expanded to various sizes depending on the application.

  These examples are presented for illustrative purposes, and readers can place test plates in other dimensions, other patterns, cross-sectional shapes (eg, rectangles, triangles), test wells with size and space, each test well It will be appreciated that other numbers of related sensors are possible and are within the scope of this disclosure.

  As best seen in FIG. 1C, in certain embodiments, the electronic test plate 100 may include a cap layer 102 to retain material in the well and reduce or prevent contamination of the material. In one configuration, the cap layer includes a patterned layer of individual caps disposed over the test well. In other configurations, the cap layer is a continuous layer extending over the top surface of the test plate. The cap layer may comprise a transparent material, for example a transparent plastic such as polycarbonate or polystyrene.

  The electronic test plate 100 shown in FIGS. 1A-1D is a structure that includes a number of material layers. The electronic test plate 100 includes a substrate 151 that mechanically supports additional layers disposed thereon. In some implementations, the substrate 151 may include glass or other optically transparent material having a thickness along the z-direction of 800 μm. Mechanical robustness sufficient to facilitate handling of the electronic test plate 100 and / or sufficient optical transparency to facilitate use of the electronic test plate with optical analysis techniques is possible. As long as other thicknesses and / or materials may be used for the substrate.

  Any portion of the electronic test plate, cap layer and test plate that contacts the material may be sterilized using common techniques such as radiation, gas, chemicals or heat sterilization, or can be serialized. May be.

  The back surface 152 may be disposed on the substrate 151 and may include a plurality of sub-layers that form an electronic device using electrical connections therebetween. In an embodiment, the back surface may have a thickness along the z direction of about 5 μm. The back surface 152 includes a sensor 120 associated with the test wells 111A-114A and a circuit 130 (eg, a switch) that accesses the sensor. In certain embodiments, the back surface 152 also includes additional electronic circuitry, such as signal processing circuitry, control circuitry, memory circuitry, and / or communications circuitry, such as wired or wireless communications circuitry described in further detail herein. May be. In certain embodiments, the back surface 152 also includes an electrical connection 135 that is arranged, for example, as an edge card connector, which is used to connect the sensors and / or other circuitry of the electronic test plate 100 to another device (eg, a host). The processor 100) is communicably connected.

  The device includes an optically clear region that can optically interrogate each well. The electronics on the back 152 are thin film electronics, such as optically transparent semiconductors and / or optically thin metals or optically transparent, to allow light to pass through the back and into the well. A conductor, for example, a thin film transistor (TFT) containing indium tin oxide (ITO) may be included. In order to facilitate the use of electronic test plates with various optical analysis techniques (eg, optical microscopes, etc.), the back surface is at least optically transparent at the interface between each test well and the back surface. In certain embodiments, the entire back surface is optically transparent. In certain embodiments, a portion of the back surface, eg, the area between the test wells, is optically opaque. In certain embodiments, the entire back surface may be optically opaque. In various configurations, the electronic test plate can be interrogated optically from either or both of the top and bottom of the well.

  For example, the test plate layer 153 has a thickness in the z direction of about 1500 μm and is disposed on the back surface 152. In certain embodiments, the material of the test plate layer 153 may include plastic or other material that can be patterned to form the test wells 111A-114A. Test wells 111 </ b> A- 114 </ b> A are defined by a well wall 160 that extends between upper electrical connection layer 154 and back surface 152. The upper electrical connection layer 154 is a conductive layer, and may include a metal (eg, gold), a metal alloy, or other conductive material. An upper electrical connection layer 154 is disposed on the test plate layer 153 and conformally coats at least a portion of the test well wall. The test plate layer 153 may have a thickness along the z direction of about 1500 μm. The top electrical connection layer may be a thin metal (eg, gold) or other conductive material. For example, the thickness of the top electrical connection along the z-axis may range from about 0.1 to about 1 μm.

  In certain embodiments, the test plate layer 153 is formed directly on the back surface 152, and this structure is referred to herein as an integrated electronic test plate. In other embodiments, the well layer 153 and the back surface are each manufactured separately and couple the well layer 153 to the back surface 152. The test well is a hydrogel (eg, 3D laminin rich gel, eg, MATRIGEL available from Corning, Inc., NY, or Gaithers, to facilitate 3D growth of cells cultured in the test well. CULTREX BME available from Trevigen, Inc. in Burg, MD. Analytes may include living cells, bacteria, viruses, fungi, microorganisms, intracellular compartments, exosomes, molecules, macromolecules, enzymes or tissue components that grow in a three-dimensional environment.

  The substance to be analyzed may include components of living cells and / or tissues, such as those that grow in a hydrogel. Tissue components may be composed of extracellular materials including proteins, saccharides, fats, carbohydrates, etc. of different cell types. For example, cancer tissue is composed of cancer cells, immune cells, nerve cells, fibroblasts, and many cell-free components. In certain embodiments, the substance to be tested may include body fluids or body fluid cells.

  The medium is specific for each substance to be tested, for example, providing the cell culture with nutrients, serum and / or antibiotics. Some examples of media are DMEM (Dulbecco's Modified Eagle's Medium), Minimum Essential Medium (MEM), and serum examples are for other biochemical needs needed to culture each cell type. In addition, fetal bovine serum (FBS).

  In certain embodiments, it may be useful to have multiple (eg, four) subpixel sensors per pixel, although more or fewer subpixel sensors are possible. The four subpixel sensors may include, for example, an impedance sensor, a chemical (pH) sensor, an acoustic sensor, and an optical sensor. FIG. 2A is a top view of a portion of the back surface 295 in the vicinity of one pixel with the four sub-pixel sensors described above. FIG. 2B is a side cross-sectional view of the test well and a portion of the back surface 295 of the test plate 290. Test well 270A includes nine pixels, three of the nine pixels are shown in this cross-sectional view, each pixel having four subpixel sensors. FIG. 2F is a top view of a test plate 290 with the test well 270A of FIG. 2B showing nine pixels.

  Referring now to FIG. 2A, the pixel 200 has four sub-pixel sensors disposed on the back surface 295. Sensors include electrical sensors such as impedance sensors, chemical sensors, acoustic sensors, and optical sensors. In this example, the impedance sensor includes interdigitated electrodes 211, 212 that facilitate the detection of the lateral impedance of the material along the x, y, and / or z directions. The chemical substance sensor includes a pH sensor and includes a TFT 221 that detects pH. The acoustic sensor includes an integrated piezoelectric sensor 231 that is used in combination with the piezoelectric ultrasonic transducer 261 and the piezoelectric film 262 shown in FIG. 2B. The optical sensor includes a photosensitive PIN diode 241. The pixel 200 also includes a TFT circuit 251 configured to facilitate access to the signal of the selected sensor.

  FIG. 2B shows a cross-sectional view of a portion of an electronic test plate that includes a test well layer 275 and a back surface 295. The test well layer 275 has a test well 270A containing a substance to be analyzed. In this example, the analysis target is a cell colony that grows in a 3D gel 298, which is a cell-free environment around the cell colony 299. 299. In this example, test well 270A is associated with nine sensor pixels on back surface 295 (see FIG. 2C). FIG. 2B shows three sensor pixels 200A, 200B, 200C each including four sub-pixel sensors similar to the sensor pixel 200 described above in a cross-sectional view. Each sensor pixel 200A, 200B, 200C includes an impedance sensor, a pH sensor, an acoustic sensor, and an optical sensor. The impedance sensors of the pixels 200A, 200B, and 200C include electrodes 271A, 271B, and 271C that mesh with each other, the pH sensor includes TFTs 221A, 221B, and 221C that detect pH, and the acoustic sensor includes a piezoelectric film ultrasonic transducer. 261 and an integrated piezoelectric sensor 231B, 231B, 231C used in combination with the piezoelectric film 262, and the optical sensor includes photosensitive PIN diodes 241A, 241B, 241C. Test well 270A is defined by test well wall 260 extending upwardly from bottom surface 265 of test well 270A. Disposed over and at least partially covering the wall surface 260 is a gold contact layer 280 that facilitates sensing impedance along the z-direction. In some embodiments, the gold contact layer 280 may be used for one or more impedance sensors of the pixels 200A-C to sense impedance along the z direction.

  As described above, an electrical impedance sensor suitable for use in the electronic test plates of the various embodiments includes an interdigitated first electrode disposed on the upper back surface and exposed to material in the test well. A second electrode may be provided. The first and second interdigitated electrodes 211, 212 (see FIGS. 2A and 2B) can be used to sense the lateral impedance in the xy plane. The impedance sensor may include a conductive contact layer, for example, a gold contact layer 280 as the third electrode. Each impedance sensor in the test well can sense a vertical impedance between the first electrode 211 and the third electrode 280 and / or between the second electrode 212 and the third electrode 280. . The arrangement of the first electrode 211, the second electrode 212, and the third electrode 280 shown in FIGS. 2A and 2B detects the electrical impedance along the x, y, and z axes in 3D. Transverse and / or vertical electrical impedance sensing can be used to determine lateral and vertical electrical impedance spectra, eg, impedance Z (ω) as a function of frequency. The measured lateral and / or vertical electrical impedance may be used to determine cell number and / or cell viability. When a cell dies, the integrity of the cell membrane, polarity, and cell morphology may change, resulting in different impedance values. Cell viability can be monitored by measuring the electrical impedance through the vertical electrodes or across the interdigitated electrodes in the lateral direction, and viability correlates with changes in impedance. is expected. In some cases, in certain cell types, a potential of 0.1 V may be applied and the magnitude of the impedance may be measured from 500 Hz to 10 kHz. In some cases, the detection limit and dynamic range includes a sensitivity of | Z | <50 ohms and θ of ˜0.50 in the frequency range of 1 KHz to 1 MHz.

  A chemical pH sensor suitable for use in the electronic test plates of various embodiments may comprise an ion sensitive silicon nitride gate of a thin film transistor. Using such a sensor, for example, the extracellular pH value can be measured. In some cases, the pH sensor can be manufactured to have a response time of less than 1 minute, a sensitivity range of pH 0.05-0.1, pH 4.0-8.0.

The acoustic emitter / sensor 262 in the test well of the electronic test plate of various embodiments is capable of 3D sensing acoustically the material in the test well. Suitable acoustic emitters / sensors are made from thin film piezoelectric sensors, such as those made from polyvinylidene fluoride (PVDF) material, or electrostrictive capacitive, such as metallized silicone, molded around the pixel. May be included. The piezoelectric emitter / sensor is configured to detect waves created by the piezoelectric emitter 261 and the piezoelectric film 262. For example, in one configuration, the piezoelectric membrane transmitter creates pulsed ultrasound that is reflected (echoed) from the interface between the cell colony 299 and the 3D gel 298. The piezoelectric sensor detects the reflected wave by amplifying a delayed echo using an amplifier (not shown) connected to the transducer 262. A time-of-flight technique for sound detection may be used to determine the location of the cell / gel interface. Motility characteristics of the cell culture by comparing the first position of the cell-gel interface detected at time t ₁ with the second position of the cell-gel interface detected at time t ₂ (Eg, degree of motion, velocity and / or acceleration) can be determined. Cell motility may be induced by creating a chemical gradient and / or by adding a chemical inducer at a predetermined distance from the cell. The acoustic sensor may have a z-position sensitivity of ± 100 μm, a pulse echo resolution of 100 nsec at an operating frequency of 1-30 MHz, and provide a z-axis permeability of 100 μm-1 cm. For example, in one embodiment, acoustic probe search may be used at pulse echo timing to continuously measure the location of vertical cell colonies.

An optical detector suitable for an electronic test plate according to various embodiments comprises at least one thin film PIN diode aligned on the back and bottom of the test well. In some embodiments, multiple thin film PIN diodes may be used per pixel, for example, about 4 may be arranged in a row. The optical detector is configured to detect light generated by, for example, a light source arranged to emit light from the top or bottom surface of the test plate through the material under test. As light flows across the test well, the light interacts with the material in the test well. For example, light emitted by a light source may be absorbed, scattered, and / or reflected by the structure of cells and / or tissues. The light that interacts with the material in the test well is detected by an optical detector and can be used to determine the optical density of the cells, and the cell number can be determined from the optical density of the cells. Further, the lateral cell motility may be determined by using the optical sensor signal and comparing the light detected at the first time with the light detected at the second time thereafter. . In some embodiments, the thin film PIN diode detector may have a sensitivity on the order of about 3000 photons, 10 ⁴ to about 10 ⁸ photons, although different illumination sources are used and other sensor sensitivities are possible. In certain implementations, the detector may have a response time of less than about 100 μsec.

  In certain embodiments, one or more oxygen sensors may be associated with the test well. This may be used to identify hypoxic cancer cells or hypoxia in the environment. Hypoxic and acidic conditions are associated with increased mutation, chromosomal destabilization, spontaneous transformation, resistance to apoptosis, and increased cellular invasion and metathesis. FIG. 2C is a top view of nine pixels 281-289 that may be associated with one test well. Pixels 281-288 are similar to pixel 200 shown in FIG. 2A and have the four sub-pixels already described. Pixel 289 includes a dissolved oxygen sensor 252 and is shown in greater detail in the plan views of FIGS. 2D and 2E, which shows pixels 288, 289, and 284 in cross-sectional view. In certain embodiments, the oxygen sensor 289 may be based on a proton exchange membrane (eg, Nafion). Nafion is a type of proton exchange membrane (also called polymer electrolyte membrane) (PEM), is semi-permeable, and is designed to conduct protons (H +). Nafion is commercially available in thin film form from Dupont. When implemented as an oxygen sensor in a test well, Nafion or other types of solid electrolytes 253 are coated on the electrodes 266-268 in the wells or are thermally adhered to the oxygen permeable membrane 252 ( For example, PTFE) is coated on top of the solid electrolyte 253. The electrodes of the oxygen sensor include a reference electrode 266, a working electrode 267, and a pair of electrodes 268. O2 dissolved in the cell culture matrix passes through the oxygen permeable membrane 252 and permeates to the solid electrolyte 253 (eg, Nafion), where O2 undergoes a reduction reaction (ie, O2 + 4H ++ 4e → 2H2O). The flow of this reduction reaction is measured by the working electrode 267 and the pair of electrodes 268, and the voltage between the reference electrode 268 and the working electrode 267 is also measured.

  FIG. 2F shows a top view of a portion of an electronic test plate 290 that includes the test well 270A of FIG. 2B with the same test wells 270B, 270C, and 270D. The back surface 295 has a repeating pattern of the same sensor pixels 200a to 200i. Each sensor pixel 200a-200i may include, for example, four sub-pixel sensors. In this example, each test well 270A-270D comprises 9 sensor pixels and 36 sensor subpixels. This configuration is given for illustration and it will be understood that an electronic test plate with more or fewer test wells associated with more or fewer pixels and sub-pixels is possible. Let's go.

As shown in FIG. 2F, the test plate has a test well pitch P- _{well 1} , where the test well pitch is the center-to-center distance between the test wells. The back side has a pixel pitch P _pixels , the pixel pitch being the center-to-center distance between pixels. As shown in FIGS. 2F-2H, the pitch of the test wells on the test plate may be different from or the same as the pitch of the back sensor pixels. FIG. 2F shows a top view of the backside with a pixel pitch P _pixel smaller than the test well pitch P _{well 1} , each test well including nine sensor pixels. FIG. 2G shows a top view of the same back with a test plate having a test well pitch P- _{well 2} smaller than P- _{well 1 and} larger than back-pitch P _pixels , each test well having four sensors. . FIG. 2H shows the same backside with the same pixel pitch P _pixel as the test well pitch P _{well 3} , each test well containing one sensor pixel. It will be appreciated that the electronic test plate need not have a constant pixel pitch or a constant test well pitch throughout the test plate. In certain embodiments, the electronic test plate may have a back surface that includes sensor pixels arranged in many different pixel pitches, and the test well on the test plate also includes many different test well pitches. May be. The width of the test well can vary from a conventional large well (eg, 5 mm) to a well width of a few hundred microns, using the same TFT back surface. For example, if a large well having a 300 μm pitch multi-detection type pixel (for example, a well having a diameter of 5 mm) is used, a small well having a 300 μm pitch multi-detection type pixel (for example, a well having a diameter of 400 μm) is used. Compared to the number of pixels per well at the time, there will be a large number of multiple sensing pixels per well.

  In some embodiments, for example, a patterned plastic can be used to fabricate the test well structure directly on top of the TFT back to create an integrated electronic test plate. In some embodiments, after the test plate is bonded to the TFT backside, the test plate and backside can be manufactured as separate structures.

  FIGS. 3A-3D are photographs of prototypes of electronic test plates fabricated using different well sizes and materials. FIG. 3A illustrates the ability to fabricate a 90 μm multi-sensing pixel circuit on the TFT backside. FIG. 3B illustrates the use of SU-8 photoresist to produce high aspect ratio wells. FIG. 3C is a photograph of a manufactured 10 μm Si microwell. FIG. 3D is a photograph of a 5 mm hydrogel test well that can be easily placed on a 3D laminin-rich gel by manual pipette use and can keep cells alive for up to 8 days in 3D embedded media. is there. In one design, test wells may be created using glass and / or injection molded plastic instead of Si, which increases the stability of the cell walls.

  Loading gels and cells into the test wells can be accomplished by pipetting the Au region (top and side walls) or passivating the Au region with a biocompatible coating such as PEG-thiol and removing the bottom of the well. It can be achieved by activating with a laminin rich gel (immersion in gel solution at 4 ° C. and spin coating at 37 ° C. before exposure to cell solution). In certain embodiments, gels may be generated in the system by polymerization within the wells. Selective delivery of gels, cells or cell culture media can be assessed by light microscopy.

  FIGS. 3E and 3F show exemplary 3D cultures of MDA-MB-231 cells on thin gold coated glass slides and silicon, respectively, which are easy to grow on gold and have a star-shaped structure. It shows that it generates.

  FIG. 4A provides a schematic diagram of an exemplary TFT subpixel sensing circuit and sensor selection circuit that can be implemented to provide sensor signals from impedance sensors, pH sensors, optical sensors, and acoustic sensors. In this example, an exemplary sensor selection circuit for a multi-sensing pixel has five query functions: vertical electrical impedance, lateral electrical impedance, local pH sensing, optical intensity, and acoustic response. .

  To interrogate the vertical electrical impedance, either Row_Imp_Select1 or Row_Imp_Select2 can be selected, for example by raising the voltage on these lines from the off state to the on state voltage (eg, the off state is approximately 5V, and the on-state voltage may be about + 15V). Increasing the voltage on these lines turns on transistor M4 or M6. One of the interdigitated electrodes is electrically connected to an external current source by M1 or M2 and the column source line I_Source + or I_Source−. Current flows through the electrode, interrogating cell, and common electrode surface (eg, gold contact layer 280 shown in FIG. 2B). The sensing electrode voltage will be sampled and buffered by the readout source follower TFT (M3 or M5) and a signal will appear on the data line (Data_Imp1 or Data_Imp2). I_source + and I_source− can be controlled by an external function generator that provides an amplitude and frequency sweep function. Since the electrode potential is buffered and amplified by the source follower M3 or M5, signal degradation and crosstalk between adjacent pixels can be reduced, for example, reduced to a negligible amount. Lock-in amplifiers can be used for excitation (I_Source) and readout (Data_Imp) to further reduce noise and interference and increase sensitivity.

  To interrogate the lateral impedance, both Row_Imp_Select1 and Row_Imp_Select2 are turned on simultaneously, and transistors M4 and M6 are turned on. The excitation current will then flow through TFT M1 to the electrode, cell, electrode, TFT M2, and then to I_source-. The voltage difference between the two interdigitated electrodes is buffered by M5 and M3 and read by transistors M4 and M6. Lateral impedance signals will appear on the data lines Data_Imp1 and Data_Imp2.

A local pH value can be detected by an ion sensitive field effect thin film transistor (pH TFT) that measures the ion concentration (eg, H ⁺ concentration in solution). The current flowing through the transistor varies as a function of ion concentration. In order to read out the pH signal on the Data_pH line, the Row M is turned on by Row_pH_light_Select while the other Row selections are kept off.

  An integrated photosensitive PIN diode D1 can be used to monitor well opacity. In order to read out the light level at the bottom of the well, the Row_pH_light_Select line can be turned on with the TFT M9 and the flow of photons can be read out with the Data_light signal line. The light sensitivity of these PIN light sensors is very good and will depend mainly on the external readout amplifier. In a well-designed system, moving in query mode can show the photosensitivity of thousands of visible photons.

  Sound detection can be achieved by a piezoelectric diode (denoted as Piezo in FIG. 4A) and a mixer diode D2 connected to a bias voltage DBias. Piezoelectric transmitters (see elements 261 and 262 in FIG. 2B) emit sonic (eg, ultrasonic) pulses that interact with structures and / or other materials in the test well. This interaction may weaken and / or otherwise alter the characteristics of the pulse as it moves through and / or around the structure and / or material. The piezoelectric sensor detects the modified acoustic pulse and in response creates an electrical signal.

  Prior to data acquisition, changes that exist for piezoelectric transducers and / or others are cleaned up by activating transistor M2. The DC component of Rbias Waveform (Dbias) then biases diode D2 with a specific range of gates, and the non-linear nature of diode D2 acts as a mixer. As a result, the received ultrasonic signal based on the sine reference signal of Rbias or the reference signal obtained by shifting the in-phase and quadrature components by 90 ° is mixed. After mixing, the resulting current is integrated over the receiver capacitance over time that is longer (eg, an integer multiple) than the time of the carrier frequency. This integrated signal is proportional to the real and imaginary parts of the received signal in the baseband that contains information about the reflectivity of the environment at a particular range of gates. These in-phase (I) and quadrature (Q) values can be read into the Data_acoustic line when Row_acoustic_select is activated.

  An acoustic sensor may be used to determine the acoustic impedance of the substance by changing the frequency, phase, and / or amplitude of the transmitted acoustic signal and sensing the resulting sound wave with the acoustic sensor. In some implementations, the time of flight of the transmitted acoustic signal may be determined.

  Further information regarding acoustic sensing applicable to the embodiments described herein is given in co-owned US Patent Application Publication No. 20130235698.

  The incident light source used for optical detection may be placed above or below the test well and can provide incident light having a controlled spectrum, pulse, width, intensity and / or collimation. 4B-4D show some configurations that can be used to provide incident light, other configurations are possible.

  FIG. 4B shows a portion of an electronic test plate 400 having test wells 401, 402 containing substances 411, 412 to be analyzed. The back surface 420 is aligned along the bottom of the test wells 401, 402. Each test well 411, 412 is associated with one or more light emitting devices 431, 432 (eg, a photodiode disposed on the back surface 420) and one or more optical sensors 441, 442. Each light emitting device 431, 432 can be energized in a synchronized manner with the selection of the optical sensors 441, 442 to provide optical sensing for the pixel or group of pixels by a sensor selection circuit. For example, the substance 411 to be analyzed will reflect at least a portion of the incident light 451 emitted by the light emitting device 431. The optical sensor 441 detects the reflected light 461 and creates an electrical signal in response.

  In certain embodiments, the incident light source for optical detection may include a source separated from the back surface that provides incident light from above or below the test well. For example, in one configuration, the incident light source may be placed over the test well, as shown in FIGS. 4C and 4D. The incident light source may provide pixelated light (shown in FIG. 4C) or non-pixelated light (shown in FIG. 4D). FIG. 4C shows an incident light source 405 positioned over the test wells 408, 409 of the electronic test plate 450. In this example, the light source 405 uses array light emitting devices 407, 408 that can be individually turned on and off to provide pixelated light and individually provide incident light to the respective wells 408, 409. The substance 411 to be analyzed may block (absorb and / or reflect) a part of the incident light 455. At least a portion of the light 455 emitted by the light emitting device 407 is transmitted to the optical sensor 441, which detects the transmitted light 465 and generates an electrical signal in response. In some embodiments, a pixelated light source may be achieved using a digital light projector (DLP) or a combination of a projector and a mirror, which reflects light from the projector, and a specific test well or test. Turn to the well group. The mirror can be connected to a moving mechanism configured to move the mirror along the x and / or y direction to provide incident light for the test well.

  In certain embodiments, as shown in FIG. 4D, incident light may be provided to the electronic test plate by a light source 415 comprising one or more light emitting devices 417 and a waveguide or light tube 416. In the example of FIG. 4D, the incident light source 415 is disposed over the test wells 403 and 404. The light emitting device 417 is optically connected to the waveguide 416 at one or more input edges of the waveguide 416. Light 419 emitted by light emitting device 417 travels along waveguide 416 by total internal reflection (TIR). Optionally, the waveguide 416 may be wedge shaped or may include an extraction feature 418 that extracts incident light 457 towards the test wells 403, 404. Optionally, one or more optical films may be deposited on the waveguide to make the incident light parallel or change the angle of the incident light. At least a portion of the incident light 457 is transmitted to the optical sensor 441, which detects the transmitted light 467 and generates an electrical signal in response. Although FIG. 4D shows a waveguide placed over the test well, in an alternative configuration, the waveguide and light emitting device may be placed under the test well, eg, on the backside. Good.

  FIG. 5A is a block diagram of an electronic test plate 500 according to some embodiments. The electronic test plate 500 includes a test plate 510 having a test well 511. The electronic test plate 500 also has a back surface 520 with sensors 521 aligned with the test well 511 such that a plurality of sensors 521 are associated with each test well 511. The back surface includes a sensor selection circuit 522, which includes, for example, a TFT switch, and the sensor selection circuit 522 gives a signal from the selected sensor at the time of parallel data output. The sensor selection circuit can be controlled by row and column selection lines and may simultaneously access multiple sensor signals.

  FIG. 5B is a block diagram of an electronic test plate 501 that is similar in some respects to the test plate 500 of FIG. 5A. The back surface 530 of the test plate 501 includes additional optional features. The electronic test plate 501 further includes a readout circuit 523 configured to receive the sensor signal of the selected sensor. The lead-out circuit 523 may optionally include a signal processing circuit 550 configured to condition the sensor signal, such as a filter, an amplifier, or the like. For example, the signal processing circuit may comprise one or more different amplifiers configured to increase the signal to noise ratio (SNR) of the sensor signal. In certain embodiments, the readout circuit may comprise an analog-to-digital converter (ADC) 524 configured to convert an analog sensor signal to a digital sensor signal. In some cases, the lead-out circuit 523 may temporarily store the digital sensor signal in the memory buffer 525. In one implementation, the back surface 530 includes a selection control circuit 526 that creates row and column selection lines, eg, in accordance with instructions received from a host processor. The electronic test plate 501 includes a communication circuit 527 configured to receive instructions from the host processor and / or send digital sensor signals to the host processor. For example, instructions from the host may include instructions regarding which sensors to access and / or the frequency of access, and these and other parameters may be determined, for example, by a user interface driven by the host processor, It may be selected by the user. The communication circuit 527 and the host processor may be configured to communicate instructions and / or data via standard protocols, such as Universal Serial Bus, IEEE 1394, ISO / IEEE 11073, or other communication protocols.

  In some embodiments, one or more electronic test plates 610 described above may be incorporated into a test system 600 as shown in the block diagram of FIG. The test system 600 includes a fluidics subsystem 620 configured to dispense material into and / or withdraw material from the test wells of the electronic test plate 610. In certain configurations, the fluidics subsystem may comprise a printer and / or other device configured to place the analyte of interest in the test well. In certain embodiments, the fluidics subsystem comprises a pipetter device configured to automatically dispense material and / or withdraw material from a test well under the direction of a controller (eg, host processor). It may be.

  The fluidics subsystem may include a functional membrane on the test plate configured to assist in loading reagents into the wells by dip coating. For example, loading gels and cells into test wells may be accomplished by passivating the Au region (upper and side walls) with PEG-thiol. Alternatively or in addition, the fluidics subsystem may include a chemical or physical surface modification of the test plate configured to allow the analyte to be adhered to the well. For example, surface modification may include activating the bottom of the well with a laminin rich gel (soaked in a gel solution at 4 ° C., spin-coated under reduced pressure, heated at 37 ° C., and cell solution A 500 μm gel layer is obtained before exposure to). Cells are suspended in a liquid phase gel at 4 ° C. and then seeded into gel-coated wells. After the gel is solidified, medium is added to allow the cells to adhere. Wash off non-adherent cells before 48 hours in culture. Selective delivery of gels, cells, cell culture media and / or chemoattractants and labels can be assessed by light microscopy.

  In some configurations, the design of surface molecules for the test plate can be achieved by reacting the test plate substrate with a piranha solution (hydrogen peroxide / sulfuric acid 2: 5 v / v) at 70-80 ° C., or Nanostrip. Reacting with 2X (Cyantek, Fremont, CA) at room temperature and drying under nitrogen, clean surface (eg gold) free of organic residues and approximately 10 ° contact with silicon A cornered hydroxyl layer is obtained. First, gold was modified with a 20 mM mixture of 11-MUA and 3-MPA alkanethiols (1:10 v / v) for 16 hours to produce a self-aligned monolayer (SAM), which was then Is exposed to a mixture of 30 mM NHS and 150 mM EDAC ester for 30 minutes. The substrate with NHS on gold is sterilized with 70% ethanol for 15 minutes and then exposed to fibronectin for 45 minutes at a concentration of 0.1 mg / ml in phosphate buffer (PBS) at room temperature. After each step of surface modification, the substrate may be rinsed with the original solvent and deionized (DI) water, respectively, to remove loosely bound portions from the surface. As a result, the immobilized fibronectin forms a robust bioadhesive layer of cell adhesive on the gold.

  In one configuration, each well is surface modified to carry biomolecules such as vitronectin, laminin, and cluster class 44 (CD44) protein or peptidomimetics and glycans, glycosaminoglycans and fats. Improve cell adhesion and cell inoculation in specific areas by chemical generation of membranes that promote cell adhesion. The sensor surface may be biochemically functionalized with extracellular tissue components, or may be physically modified to create a surface shape that affects cell function in a targeted manner. . For example, 3D gels can be made from hyaluronan molecules that are rich in invasive cell populations among the populations of cancer cells that affect sensor signals. To improve many parameters such as gel adhesion, cell function, mechanical scaffolding, ultimately signal-to-noise ratio, nanomaterials (eg, containing biologically targeted moieties, or No nanowires, nanoparticles, nanotubes, nanorods) may be used on the surface of the test plate and / or 3D gel.

  In certain embodiments, a hybrid matrix may be created. These hybrid matrices may include at least one of nanomaterials and thermoresponsive 3D gels. Matrix components may be premixed at a specific ratio and delivered to each well by pipette, path or printer nozzle. Alternatively, the matrix components may be delivered from different nozzles and mixed in each well. The fluidics subsystems may be arranged to provide the ability to change and adjust the properties of the matrix of each cell type. In some scenarios, mechanically strong metal particles or rods may be added to adjust the properties of different cell type matrices. In one scenario, the gel may be premixed with the nanomaterial to induce different properties.

  In certain embodiments, one or more of the electronic test plates and / or fluidics subsystems may be connected to the host processor 630. In some configurations, the host processor 630 can control material dispensing and / or withdrawal from the test well / material to the test well and control the type of substance detected and / or the frequency of detection. be able to. In some configurations, the host processor may be configured to analyze sensor signals and provide processor output (eg, formatted as a report that can be printed or displayed on a display). . The host processor may analyze two or more of the detected signals together and / or use information from one detected signal and analyze another detected signal.

  In some scenarios, the electronic test plate may include electronic circuitry (eg, a processor) configured to provide some or all of the sensor signal control and / or analysis. In other embodiments, the electronic test plate may transmit the sensor signal to an external processor for performing the analysis, for example, in analog or digital form. The electronic test plate may be configured to include wired or wireless communication circuitry to transmit data and control signals and / or other information to / from the host processor / other information from the host processor. Good.

  Analysis of the sensor signal by the processor will yield an output that includes one or more characteristics of the substance to be analyzed. Non-limiting sets of material features include lateral and vertical impedance, optical spectrum, phenotypic signature, chemical signature, functional signature, acoustic signature, mechanical signature, generated oxygen, cellular Attachment and diffusion, cell proliferation, cell signal transduction, toxicity, cell electroporation, cell location, cell number, cell viability, cell stiffness, matrix (gel) stiffness, extracellular pH, motility, There may be lateral and / or vertical mass transfer, response to therapeutic agents, response to environmental challenges, and / or behavior directed towards the cytoskeleton of interest.

  Processor and data analysis software, eg MatLab (as a rapid prototyping language), principal component analysis (as a traditional data analysis method), to test the significance of group differences (eg of cell populations) Analysis of sensor signals by linear regression (eg, impedance) to predict response variables as a function of explanatory variables (time, temperature, etc.), including one or more characteristics of the substance being analyzed An output will be obtained and a new variety of signatures could be captured that are not equal to the sum of two or more characteristics of the substance to be analyzed. In certain embodiments that maximize the number of test wells or continuous monitoring time, high-throughput analysis of the data could identify new patterns and signatures.

  Sensor signal analysis enables stratification of cancer cell colonies by invasiveness, structure and growth, giving a signature that progresses from normal to metastatic cancer disease, or multi-detector measurement panel The characteristics of the substance from can be predicted. In certain embodiments, some or all of the test wells are exposed to an analyte, such as a drug or toxicant (eg, environmental, chemical or biological venom), and the cellular response to the analyte is determined continuously. And / or in parallel.

  In certain embodiments, analyzing the sensor signal may include determining an optical spectrum of the material. For example, a cell or tissue component may be optically characterized by switching the incident light source toward the test well to several wavelengths (eg, red, green and blue) and measuring the optical response. For example, the intensity of light reflected by the substance to be analyzed or passed through the substance to be analyzed can be measured over the entire wavelength spectrum of incident light using an optical sensor (eg, a PIN photodiode) associated with the test well. Also good. The incident light source and optical sensor of the electronic test plate are used in addition to or instead of, and based on the absorption, transmission and / or reflection of light of the incident light source by the substance, the position, movement and / or morphology of the cells May be measured. An image analysis tool (eg, position and / or edge algorithm) may be applied to the output of the photodiodes in the photodiode array to determine the position of the cells in the well.

  By analyzing the signal from one or more optical sensors associated with the test well, cell movement and / or morphology may be determined based on the amount of incident light absorbed and / or reflected by the analyte. Good. In addition or alternatively, cell movement and / or morphology may be determined based on the acoustic signature of the substance. The acoustic response of the substance under test changes with cell movement and / or changes with changes in cell or tissue morphology. The acoustic response may include the reflected intensity, frequency and / or phase of the acoustic signal and may be used to derive mechanical properties of the substance to be measured, eg stiffness, mass, cell distribution .

  In addition or alternatively, cell movement and / or changes in cell morphology may be detected based on impedance sensing. In some implementations, multiple sensors may be used to detect cell movement. For example, the acoustic, impedance, and / or optical signatures of a substance may be compared with one or more known signatures or signatures already obtained to determine changes in cell movement and / or morphology. In addition or alternatively, microscopic images may be used to measure or confirm cell movement and morphology.

  Cell viability may be determined based on, for example, the impedance, morphology and / or acoustic signature of the substance. When cell death occurs, the cell impedance, morphology, and acoustic signature change. The known signature of the surviving cells may be compared with a signature taken later to determine whether the cells remain viable or how many cells remain viable.

  In certain embodiments, in addition or alternatively, a test plate chemical sensor may be used to determine cell viability. Cell death will be indicated when the pH of the cell drops from normal to less than 7. In addition or alternatively, the ability to determine or confirm cell viability using a LIVE / DEAD Viability / Cytotoxicity cell-impertain stains that uses only damaged or damaged cell membranes with a microscope. Good.

  The mechanical properties and / or morphology of motile cells can be distinguished from cellular properties and / or morphology, and these changes can be detected by analyzing the acoustic response of the substance under test. By using the acoustic sensor, it is possible to measure the vertical and lateral directions of the cell colony position and the movement of the cells in the vertical and lateral directions. By using acoustic sensors in parallel, the mechanical properties of the cells, the vertical and lateral positions of the colonies, and the movement of the cells in the vertical and lateral directions can be measured with high throughput.

  The processor may be configured to compare signals from adjacent wells that differ only in one aspect. Such an analysis can provide differential information that allows a high level of common aspects of noise rejection. For example, acoustic echoes from various interfaces can be accurately subtracted from signals derived from different wells only in the presence or absence of cells. Reflection from the wall is generally common to the two wells and can be subtracted. In a similar manner, signals from the same well taken at different times can be subtracted from each other, giving only a time-change perspective.

  Some embodiments include using large (eg, 5 mm diameter) test wells and utilizing large numbers of multi-sensing pixels. Using large diameter wells and many pixels yields many signatures to extract cell location, viability and / or other features. Cell location, viability and / or other characteristics may be calibrated with a known initial number (eg, 20K, 10K and 5K wells) and known viability (eg, greater than 85% per well). Lateral and vertical motility may be measured at a predetermined distance by combining the data from optical, acoustic and electrical impedance measurements after 24 hours of incubation. The motility index may be confirmed or determined from these measurement results with increased accuracy according to the correlation with the measurement of the optical microscope.

  A phenotypic signature is a collection of multiple intracellular processes that involve the expression of genes and proteins that result in the sophistication of specific morphology and function of the cell. The phenotypic signature can be determined using electronic test plate sensors based on cell morphology, cell 3D structure and motility.

  In certain embodiments, one or more electronic test plates, fluidics subsystems, and / or dispenseable materials may be placed in an incubator 640 (eg, a portable incubator) that provides a controlled test environment. In one configuration, parallel real-time detection and phenotyping can be performed using the system shown in FIG. The environmental parameters of the incubator 640 are controlled by the host processor 630 and may provide an environment tailored to the tests being performed.

  FIG. 7 is a flow diagram illustrating a method according to an embodiment. The multiple characteristics of the substance to be analyzed is that multiple sensors detect over time using multiple sensors arranged in association with each well of the test plate containing the substance. (710). At least one of the plurality of sensors associated with the well is configured to detect a characteristic of the material that is different from a characteristic detected by another of the plurality of sensors associated with the test well. An electrical sensor signal is generated based on the sensed feature (720). Activate the selection line (730) and access the signal of one or more selected sensors such that the sensor signal of the selected sensor is provided at the data output (740). According to certain embodiments, providing sensor signals includes providing sensor signals simultaneously on parallel data buses. Substances may be monitored substantially continuously during the test protocol using the method outlined in FIG.

  In certain embodiments, the sensor signal can be adjusted by filtering and / or amplification. When using an amplifier to tune the sensor signal, it may be useful to use a differential amplifier that provides a common mode of rejection that increases the signal to noise ratio of the amplified sensor signal. As described above, detecting a plurality of features of a substance may include detecting at least one feature in a plurality of dimensions.

Claims (10)

A device,
A test plate having a plurality of wells, each well configured to contain a substance to be analyzed;
Configured to detect a characteristic of the substance and generate a sensor signal based on the detected characteristic, wherein a plurality of sensors are arranged to be associated with respective wells, and at least one sensor of the plurality of sensors is A sensor configured to detect a characteristic of a substance that is different from a characteristic detected by another sensor of the plurality of sensors;
A sensor selection circuit connected to the sensor and arranged on a back surface disposed along the test plate and configured to allow a sensor signal of the selected sensor to be accessed at a data output. .   The device of claim 1, wherein the device has one or more optically transparent regions that can optically interrogate each well.   The device of claim 1, wherein at least one of the sensor selection circuit and the sensor comprises a thin film transistor (TFT).   The device of claim 1, wherein the plurality of sensors comprises two or more of an electrical sensor, a chemical sensor, an optical sensor, an acoustic sensor, and an oxygen sensor.   Said sensor signal is impedance, optical spectrum, phenotypic signature, biophysics signature, chemical signature, functional signature, mechanical signature, cell movement, cell attachment and diffusion, cell invasion, cell proliferation, Cell signal transduction, cell pathway, toxicity, cell electroporation, cell location, cell number, cell viability, cell stiffness, matrix stiffness, extracellular pH, motility, lateral mass transfer and The device of claim 1, comprising information about one or more of vertical mass transfer, behavior directed to the cytoskeleton to be analyzed. A system,
A test plate having a plurality of wells, each well configured to contain a substance to be analyzed;
Configured to detect a characteristic of the substance and generate a sensor signal based on the detected characteristic, wherein a plurality of sensors are arranged to be associated with respective wells, and at least one sensor of the plurality of sensors is A sensor configured to detect a characteristic of a substance that is different from a characteristic detected by another sensor of the plurality of sensors;
A sensor selection circuit connected to the sensor and arranged on a back surface disposed along the test plate and configured to allow a sensor signal of the selected sensor to be accessed at a data output;
A readout circuit configured to receive and process the selected sensor signal present in the data output. A method of manufacturing a device comprising:
Creating a test plate having a plurality of wells, each well configured to contain a substance to be analyzed;
Manufacturing a plurality of sensors configured to detect characteristics of the substance and create a sensor signal based on the detected characteristics;
Manufacturing a sensor selection circuit connected to the sensor and configured to allow a sensor signal of the selected sensor to be accessed with a data output;
Arranging the sensors relative to the well such that a plurality of sensors are associated with each well, each of the plurality of sensors being different from a feature detected by another sensor of the plurality of sensors. Aligning associated with a well configured to sense a characteristic of the material. Using a plurality of sensors associated with each well to detect a plurality of characteristics of a substance to be analyzed disposed in a well of a test plate over time, wherein at least one of the plurality of sensors is Detecting, configured to detect a characteristic of the substance that is different from a characteristic detected by another of the plurality of sensors;
Creating a sensor signal based on the detected feature;
Activating the address line and making the sensor signal of the selected sensor accessible at the data output. A device,
A test plate having a plurality of wells, each well containing a substance to be analyzed;
A device comprising an acoustic sensor connected to each well.   A sensor selection circuit connected to the acoustic sensor and arranged on a back surface disposed along the test plate and configured to allow a sensor signal of the selected sensor to be accessed at a data output; The device of claim 9.

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